The goals of the proposed research are to elucidate mechanisms of T-cell signal transduction through the cell membrane by the TCR/CD3 complex, and from the cytoplasm to the nucleus by the nuclear factor of activated T cells, NFAT, as regulated by the phosphatase calcineurin and by other interacting proteins. These mechanisms are crucial for T-cell activation, T-cell function, immune suppression and general T-cell biology. The research will pursue two specific aims. Specific Aim 1 is to elucidate the topology of the T-cell receptor complex. While structures of the ectodomain components are known, the topology of the complex and the mechanisms of signal transduction are still unclear. We propose to design systems that enable mapping of domain interfaces and determining the TCR/CD3 topology. This includes producing CD3 and TCR ectodomains anchored in membrane mimics, such as micelles and phospholipids nanodiscs. We will create e? single chain constructs and introduce paramagnetic sites to enable domain-domain contact measurements with NMR and EPR methods. To achieve this we will link chains and tags using a sortase approach. The proposed research will lead to structures of full-length TCR/CD3 components including the transmembrane regions and eventually to complete structures of the TCR/CD3 complex. It will facilitate understanding of the mechanism of TCR/CD3 signal transduction. Specific Aim 2 is to elucidate mechanisms that trigger nuclear translocation of NFAT. It will focus on NFAT's N-terminal regulatory domain, which contains the transactivation domain, numerous proline-rich phosphorylation sites and a nuclear localization sequence. It is primarily unstructured alone but adopts some structure when bound to the phosphatase calcineurin (Cn), to the regulatory protein Homer, to other proteins of little known function, and to transcriptional co-activator proteins in the nucleus. Despite the importance of this domain little is known about its mechanisms of function. Here we will elucidate elements of local structure and structural changes upon phosphorylation or dephosphorylation in NFAT's regulatory domain. We will determine structures of complexes with the Homer3 scaffolding protein and the transcriptional co-activator ARC105/Med15. We will study the functional relevance of these interactions. Finally, we will develop small-molecule inhibitors of the calcineurin/NFAT interaction to create immune-suppressive agents that specifically prevent NFAT dephosphorylation but will not affect calcineurin's general enzymatic activity. These new agents have the promise to avoid the long-term toxic side effects of traditional immune-suppressive drugs. The expected outcome of this research will be a better understanding of the mechanisms that regulate NFAT function triggering the immune response. In addition, new first-of-kind small molecule immune-suppressive agents will be developed.